journal of medicine
The
established in 1812
vol. 362
no. 8
A bs t r ac t
Background
Stuttering is a disorder of unknown cause characterized by repetitions, prolongations, and interruptions in the flow of speech. Genetic factors have been implicated
in this disorder, and previous studies of stuttering have identified linkage to markers on chromosome 12.
Methods
We identified a missense mutation in the N-acetylglucosamine-1-phosphate transferase gene (GNPTAB), which encodes the alpha and beta catalytic subunits of GlcNAcphosphotransferase (GNPT [EC 2.7.8.15]), that was associated with stuttering in a
large, consanguineous Pakistani family. This mutation occurred in the affected
members of approximately 10% of Pakistani families studied, but it occurred only
once in 192 chromosomes from unaffected, unrelated Pakistani control subjects
and was not observed in 552 chromosomes from unaffected, unrelated North
American control subjects. This and three other mutations in GNPTAB occurred in
unrelated subjects with stuttering but not in control subjects. We also identified
three mutations in the GNPTG gene, which encodes the gamma subunit of GNPT, in
affected subjects of Asian and European descent but not in control subjects. Furthermore, we identified three mutations in the NAGPA gene, which encodes the
so-called uncovering enzyme, in other affected subjects but not in control subjects.
These genes encode enzymes that generate the mannose-6-phosphate signal, which
directs a diverse group of hydrolases to the lysosome. Deficits in this system are
associated with the mucolipidoses, rare lysosomal storage disorders that are most
commonly associated with bone, connective tissue, and neurologic symptoms.
Conclusions
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Me thods
We analyzed families that had participated in previous linkage studies12 and focused on the largest family, designated PKST72 (Fig. 1). In addition, we studied unrelated cases of stuttering in
46 Pakistani subjects (one from each of the previously studied families12) and 77 additional unrelated persons with stuttering from Pakistan, as
well as 270 affected, unrelated persons from the
United States and England. This last group was
enrolled through public appeal, and potential
participants were required to meet the following
criteria on screening: age of 8 years or older; stuttering duration of 6 months or longer; evidence
of a family history of stuttering; and speech characterized by more than 4% stuttering dysfluencies,
as measured with the Stuttering Severity Instrument, 3rd edition (SSI-3),15 or a well-characterized
standard reading passage.16 The mean and the
overall distributions of dysfluency scores have
been shown to be similar with the use of these
two tests,15,16 and the distribution of scores with
the use of these two instruments in our population of stuttering subjects was similar as well
(see Fig. 1 in the Supplementary Appendix, available with the full text of this article at NEJM.org).
Young children, in whom recovery from stuttering is common, were excluded from the study;
also excluded were subjects who reported neurologic or psychiatric symptoms.
The ancestry of the Pakistani and North
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m e dic i n e
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17
16
6
27
Ga
40
18
Ga
28
29
42
Ga
43
41
73
Ga
74
Ga
75
aa
76
aa
11
30
Ga
44
77
aa
78
aa
20
45
79
aa
47
Ga
13
33
15
48
34
49
84
85
Ga
24
35
50
51
Ga
64
Ga
82 83
Ga GG
14
23
63
81
aa
22
32
46
80
aa
12
21
31
62
72
Ga
10
19
52
GG
65
GG
86 87 88
Ga GG Ga
53
66
Ga
67
Ga
89
aa
54
Ga
55
GG
68
Ga
56
Ga
69
GG
Genotype
25
36
91
aa
92
aa
93
aa
94
95
aa
96
aa
97
aa
98
99
37
57
Ga
58
GG
70
71
Ga
38
59
39
60
61
No. of
Unaffected
Subjects
No. of
Affected
Subjects
4
9
2
3
13
12
GG (Glu/Glu)
Ga (Glu/Lys)
aa (Lys/Lys)
90
Ga
26
100 101
Line
Combo
4-C
H/T
36p6
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The
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The variant showing the highest degree of cosegregation with stuttering in Family PKST72 was a
mutation (G3598A) that predicts the substitution
of a lysine residue for a glutamic acid residue at
position 1200 (Glu1200Lys) in GlcNAc-phosphotransferase (encoded by GNPTAB) (Fig. 2). With
three exceptions, the affected persons in Family
PKST72 carried one or two copies of this variant.
Further inspection of the affected noncarriers
showed that they carry common alleles at the adjacent marker D12S1607, whereas the 25 affected
family members carried either one or two copies
of the least common allele of D12S1607 in this
population (prevalence, 0.03). This suggests that
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GNPTAB
GNPTAB c.961AG
(p.Ser321Gly)
Homo sapiens
Macaca mulatta
Canis lupus
Bos taurus
Mus musculus
Rattus norvegicus
Gallus gallus
Danio rerio
GNPTG
GNPTAB c.1363GT
(p.Ala455Ser)
CTGCCNGTCGT
321
SKQDEDISASRFEDNEELRY
SKQDEDISASRFEDNEELRY
SKQDEDVSASRFEDNEELRY
SKQDEDVSASRFEDNEELRY
SKQDEDVSASRFEDNEELRY
SKQDEDVSASRFEDNEELRY
SKQDEDISASRFEDNEELRY
SKQDEDVSASRFEDNEELRY
ACAAGNCTTGT
455
IKDGYCDKACNNSACDWDGG
IKDGYCDKACNNSACDWDGG
VKDGYCDKACNNSACDWDGG
IKDGYCDKACNNSACDWDGG
IKDGYCDKACNNSACDWDGG
IKDGYCDKACNNSACDWDGG
IKDGYCDKACNNSACDWDGG
IKDGYCDKACNNSACDWDGG
GNPTG c.11_19dup
(p.Leu5_Arg7dup)
Homo sapiens
Pan troglodytes
Rattus norvegicus
Mus musculus
Gallus gallus
NAGPA
TGCAGNGAAGA
74
AGGPAPAGAAKMKVVEEPNA
ARGPAPAGAAKMKVVEEPNS
AQGPAPTHAGKMKVVEEPNT
SQGPAPACAGKMKVVEEPNT
AALGVLASAGKMKIVEEPNT
NAGPA c.252CG
(p.His84Gln)
Homo sapiens
Macaca mulatta
Bos taurus
Rattus norvegicus
Mus musculus
Gallus gallus
GAGTTNAAAAT
624
NLTFQNTNDEEFKMQIFVEV
NLTFQRTNDEEFKIQITVEV
NLTFQNTNDEEFKIQITVEV
NLTLQGKNDEEFKIQIVVEV
NLTLQNANDEEFKIQIAVEV
NLTLQNSNDEEFKIQIAVEV
NLTFLNKNDEEFKMQVAVEV
NITFQSTDHHDFIMTFSVSV
GNPTG c.74CA
(p.Ala25Glu)
AGCCNCGCCNN
5
MAAGLARLLLLLGLSAGGPA
MAAGLARLLLLLGLSARGPA
MAGRLTGFLMLLGLAAQGPA
MAGRLAGFLMLLGLASQGPA
..MAAARLLLAVFVGAALGV
AACCCNGCTGC
328
STVVCVHEPRCQPPDCHGHG
STVVCVHEPRCQPPDCHGHG
STVVCVHEPRCQPPDCSGHG
STVVCVHEPRCQPPNCSGHG
STVVCVHEPRCQPPDCSGHG
STIVCVHEPACEPADCSGHG
TGCAGNAATGG
1200
RFLHMHELQEWRAYRDKLKF
RFLHMHELQEWRAYRDKLKF
RFLHMHELQEWRAYRDKLKF
RFLHMHELQEWRAYRDKLKF
RFLHMHELQEWRAYRDKLKF
RFLHMHELQEWRAYRDKLKF
RFLHMHELQEWRAYRDKLKF
RFLHMTELQEWRIYRDKLKF
GNPTG c.688CG
(p.Leu230Val)
CCCAGNTGGAG
688
KTPEENEPTQLEGGPDSLGF
KTPEENEPTQLEGGPDSLGF
KVPGETHPTQLAGDSKGLGL
KVPGETHPTQLAGGSKGLGL
KATEEKE.AEKQNMKTSLQF
NAGPA c.982CT
(p.Arg328Cys)
TCGCANTTCAG
84
GLAVRTFVSHFRDRAVAGHL
GLAVRTFVSHFRDRAVTGHL
RPSVRTFVSYFADRAVPGHL
RAAVRTFVSHFEGRAVAGHL
HAAVRTFVSHFEGRAVAGHL
RCCTRTFVSYVRRRAVYGHF
GNPTAB c.3598GA
(p.Glu1200Lys)
GNPTAB c.1875CG
(p.Phe624Leu)
NAGPA c.1538_1553del
(p.Phe513SerfsX113)
CCCCTNNANNN
513
GEPLAAEKEQPGGAHNPFKD
GELLAVEKEQPGGAHNPFKD
GEHPAAEKEQLGDSSNPFKD
GDALAAEKEQTEETCNPFKD
GEALTAEKEHMEETSNPFKD
GCA.................
JOB: 36208
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Gene
Mutation
Change in
Amino Acid
Pakistani
Pakistani
Case Subjects Controls
(N = 123)
(N = 96)
North American
British
North American
Case Subjects
Controls
(N = 270)
(N = 276)
c.961AG
p.Ser321Gly
Exon 11
c.1363GT
p.Ala455Ser
Exon 13
c.1875CG
p.Phe624Leu
Exon 19
c.3598GA
p.Glu1200Lys
c.11_19dup
p.Leu5_Arg7dup
GNPTG
Exon 1
Exon 2
c.74CA
p.Ala25Glu
Exon 9
c.688CG
p.Leu230Val
Exon 2
c.252CG
p.His84Gln
Exon 6
c.982CT
p.Arg328Cys
p.Phe513SerfsX113
NAGPA
Exon 10 c.1538_1553del
* GNPTAB denotes the N-acetylglucosamine-1-phosphate transferase gene for the alpha and beta subunits, GNPTG the
N-acetylglucosamine-1-phosphate transferase gene for the gamma subunit, and NAGPA the N-acetylglucosamine-1phosphodiester alpha-N-acetylglucosaminidase gene.
Because the affected cases in our study were ascertained on the basis of having nonsyndromic
stuttering, we interviewed and carried out a more
detailed clinical examination of three affected
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683
The
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secondary to the developmental delay that is typical in these disorders, but the sparing of some
intellectual functions in mucolipidosis type III25
suggests that speech deficits may be primary
rather than secondary in this disorder.
There may be several reasons why the mutations we identified in GNPTAB and GNPTG result
in stuttering rather than in mucolipidosis types
II and III. First, mucolipidosis types II and III are
generally believed to be autosomal recessive disorders, and all the unrelated affected persons in
our sample, with two exceptions, were heterozygous and thus not expected to have either of these
disorders. Another possibility is that all but one
of the mutations we identified are missense
rather than protein-truncation or deletion mutations, which are typically observed in mucolipidosis types II and III, and presumably have a
more severe effect on protein function. None of
the mutations we observed in GNPTAB and GNPTG
have been identified in persons with mucolipidosis type II or type III.27-30
No human disorder has yet been associated
with mutations in NAGPA. This could be viewed as
surprising, given that such mutations are predicted to have an effect on many different lysosomal
enzymes. Our study suggests that a primary consequence of NAGPA mutation is nonsyndromic,
persistent developmental stuttering.
An important reason to investigate stuttering
is to better understand the neural structures and
functions within the brain that generate human
speech, which are poorly understood. Data regarding expression of these three genes in the
human brain is limited. Data for the mouse brain
are available for NAGPA and GNPTG. GNPTG has
the most localized expression in the brain, with
high levels of expression in the hippocampus,
hippocampal formation, and cerebellum (accord-
of
m e dic i n e
References
1. Bloodstein O, Ratner NB. A handbook
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cal and offspring analyses of developmental speech disorders using data from the
Colorado Adoption Project. J Speech Lang
Hear Res 1997;40:778-91.
8. Bloodstein O. Stuttering in families of
adopted stutterers. J Speech Hear Disord
1961;26:395-6.
9. McFarlane WB, Hanson M, Walton W,
Mellon CD. Stuttering in five generations
of a single family: a preliminary report
including evidence supporting a sex-modified mode of transmission. J Fluency Disord 1991;16:117-23.
zen FH. Combined tarsal and carpal tunnel syndrome in mucolipidosis type III:
a case study and review. Ann N Y Acad Sci
2009;1151:77-84.
27. Bargal R, Zeigler M, Abu-Libdeh B, et
al. When mucolipidosis III meets mucolipidosis II: GNPTA gene mutations in 24
patients. Mol Genet Metab 2006;88:35963. [Erratum, Mol Genet Metab 2007;91:
299.]
28. Kudo M, Brem MS, Canfield WM.
Mucolipidosis II (I-cell disease) and mucolipidosis IIIA (classical pseudo-Hurler
polydystrophy) are caused by mutations in
the GlcNAc-phosphotransferase /-subunits precursor gene. Am J Hum Genet
2006;78:451-63.
29. Raas-Rothschild A, Bargal R, Goldman
O, et al. Genomic organization of the
UDP-N-acetylglucosamine-1-phosphotransferase gamma subunit (GNPTAG) and its
mutations in mucolipidosis III. J Med
Genet 2004;41(4):e52.
30. Tiede S, Muschol N, Reutter G, Cantz
M, Ullrich K, Braulke T. Missense mutations in N-acetylglucosamine-1-phosphotransferase / subunit gene in a patient
with mucolipidosis III and a mild clinical
phenotype. Am J Med Genet A 2005;137A:
235-40.
31. Allen Institute for Brain Science Web
site. (Accessed January 29, 2010, at http://
mouse.brain-map.org/brain/Gnptg.
html?ispopup-1.)
Copyright 2010 Massachusetts Medical Society.
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